Identification of Pinus elliottii var. elliottii X P. caribaea var. hondurensis hybrids using the chloroplast trnL-F intergenic Spacer

Breeding programs based on hybrids of tree species often have a need to unambiguously distinguish intraspecific and self progeny from hybrid progeny. The interspecific hybrid of P. elliottii var. elliottii (PEE) and P. caribaea var. hondurensis (PCH) is difficult to reliably distinguish from pure PEE based on morphology, especially at a young age. We examined the trnL-F intergenic spacer region of the paternally inherited chloroplast genome for a polymorphism that may distinguish these two taxa. Sequencing this region indicated there were two haplotypes, one which was specific to PEE, designated (+), the other designated (–), was shared by both taxa. This result was consistent with other studies which suggest past introgression of PCH into PEE. A PCR assay was developed to detect the PEE specific haplotype. This haplotype was found at a frequency of 0.6 in a sample of 22 PEE from a breeding population but was absent from a sample of 30 PCH parents. As expected, hybrids from crosses of PCH pollen donors and maternal (+) haplotype PEE had (–) haplotypes. In situations where the mother can be genotyped, and the pollen pool consists of maternal (self) or PCH pollen, this assay would unambiguously determine the hybrid status of offspring from 60 % of the PEE parents tested. Where paternal candidates include other PEE, the assay would conclusively identify non-hybrid progeny at a lower percentage determined by the frequency of the (+) haplotype in the pollen pool. The chloroplast trnL-F spacer assay would be useful for verifying hybrids from future seed orchards based on mother trees with (+) haplotype or for (+) haplotype mother trees in current orchards. The assay will also confirm suspected pure PEE individuals where they have a (+) haplotype but will be inconclusive where they have a (–) haplotype.